Our understanding of gene networks that allow fungal pathogens to successfully interpret and respond to the host environment is rudimentary, and lags behind the more sophisticated knowledge of regulatory circuits that govern virulence of bacterial and viral pathogens. We seek to exploit tools we have developed in the human fungal pathogen Cryptococcus neoformans to approach this question. C. neoformans is an opportunistic yeast pathogen responsible for over 1 million infections and 600,000 deaths annually. In published studies, we identified several transcriptional regulators that control pathogen fitness in infected mice. Pivotal among these is the Gat201 GATA family protein that controls virulence, capsule formation, and the ability to inhibit phagocytosis. We recently determined that Gat201 controls the response of ~16% of the C. neoformans genome to environmental signals. To identify its direct targets we have developed chromatin immunoprecipitation-microarray (ChIP-chip) methods for C. neoformans. Among the genes that are directly bound and regulated by Gat201 are seven transcription factors, suggesting the existence of a transcriptional network. Two of these regulators (Liv3 and Cir1) have been implicated in pathogenicity and we have recently demonstrated that another, Gat204, controls fitness during infection of mice and the inhibition of phagocytosis in vitro. These data suggest that C. neoformans implements a network involving at least four regulators to control virulence. We seek to understand how this network functions. Characterizing targets of this network additionally provides an opportunity to identify novel virulence mechanisms. We seek to identify the mechanisms the network must activate to promote disease. Thus, our aims are as follows: (1) Determine the architecture and functional properties of the Gat201 virulence network. We will define the direct and indirect transcriptional targets of each regulator. Analysis of the network architecture is expected to yield testable predictions that could not be inferred from the study of individual regulators. In parallel, we will determine whether these transcriptional regulators have similar or distinct roles in infection. By delineating the functions of the key transcription factors that control virulence and their relationships to each other and target genes, these studies are anticipated to reveal how regulatory factors collaborate to control the virulence properties of the pathogen. (2) Exploit the regulatory network to identify novel capsule-independent virulence mechanisms. Intriguingly, Gat201 activates the expression of genes coding for three enzymes involved in chitin synthesis. Thus, we will test the hypothesis that Gat201 activates the production of chitin and/or chitin-derived polysaccharides and that these are necessary for C. neoformans to inhibit phagocytosis by macrophages thereby promoting virulence. Should we disprove this hypothesis, an alternative approach will be taken in which we test knockouts in targets of the regulatory network. These studies are anticipated to reveal novel mechanisms whose activation by the network is necessary for pathogen success. PUBLIC HEALTH RELEVANCE: The diagnosis and treatment of serious fungal infections in humans is a major unmet clinical challenge. Cryptococcus neoformans is among the most important fugal pathogens, accounting for approximately one-third of worldwide deaths from HIV/AIDS. This study, if successful, will define novel mechanisms by which this pathogen causes disease and thereby providing new targets for the development of therapeutics.